Abstract

The genome of pancreatic ductal adenocarcinoma (PDAC) frequently contains deletions of tumour suppressor gene loci, most notably SMAD4, which is homozygously deleted in nearly one-third of cases. As loss of neighbouring housekeeping genes can confer collateral lethality, we sought to determine whether loss of the metabolic gene malic enzyme 2 (ME2) in the SMAD4 locus would create cancer-specific metabolic vulnerability upon targeting of its paralogous isoform ME3. The mitochondrial malic enzymes (ME2 and ME3) are oxidative decarboxylases that catalyse the conversion of malate to pyruvate and are essential for NADPH regeneration and reactive oxygen species homeostasis. Here we show that ME3 depletion selectively kills ME2-null PDAC cells in a manner consistent with an essential function for ME3 in ME2-null cancer cells. Mechanistically, integrated metabolomic and molecular investigation of cells deficient in mitochondrial malic enzymes revealed diminished NADPH production and consequent high levels of reactive oxygen species. These changes activate AMP activated protein kinase (AMPK), which in turn directly suppresses sterol regulatory element-binding protein 1 (SREBP1)-directed transcription of its direct targets including the BCAT2 branched-chain amino acid transaminase 2) gene. BCAT2 catalyses the transfer of the amino group from branched-chain amino acids to α-ketoglutarate (α-KG) thereby regenerating glutamate, which functions in part to support de novo nucleotide synthesis. Thus, mitochondrial malic enzyme deficiency, which results in impaired NADPH production, provides a prime 'collateral lethality' therapeutic strategy for the treatment of a substantial fraction of patients diagnosed with this intractable disease.

a, Schematic of the first enzymatic step of BCAA catabolism to branched chain ketoacid (BCKA). b, Time course of expression of BCAT2, ME3, pAMPKα-T172 and total AMPK following ME3 depletion in PATU8988T cells. c, Expression of BCAT2 and ME3 in cells treated with another independent ishRNA (ishME3#3). d, Expression of BCAT2 and ME3 in BxPC3 cells. e, Expression of BCAT2 and ME3 upon depletion of ME1 and ME3 using independent (non-dox dependent) shRNAs. f, Expression of ME3 and BCAT2 upon siRNA depletion of ME3 in PDAC lines. β-Actin used as loading control. g, h, Raw flow cytometry data of DCFDA-stained cells for measurement of ROS in PATU8988T (g) and BxPC3 (h) cells upon ME3 depletion. i, Raw flow cytometry data of MitoSOX staining in PATU8988T and ME3-depleted PATU8988T cells. Antimycin A used as positive control. j, Immunoblot showing time course of expression of NRF2 in cells with dox-induced ME3 depletion. β-Actin used as loading control. k, IHC images showing NRF2 staining in ME3-depleted and control xenograft tumours (Scale bar= 50μm).